The recent emergence of novel molecular fluorescent probes and proteins has enabled the in-vivo interrogation of molecular function and significantly enhanced our ability to study pathogenesis in vivo. While advances in fluorochrome probe development have been rapid, the corresponding technologies for in-vivo imaging have been largely limited to qualitative photographic approaches of close to the surface events (reflectance imaging). In order to image molecular events deeper in tissue, we have recently developed a prototype scanner for fluorescence-mediated molecular tomography (FMT) of small animals and imaging of molecular targets. Major challenges that remain in vivo are a) to improve resolution, b) to quantitatively image the full-body distribution of specific molecular markers and c) to merge molecular reconstructions with high-resolution surface features for high positional accuracy rendering. In this proposal we seek to develop a highly optimized imaging system with unprecedented three-dimensional detection capacity. In particular we propose the use of a time-gated intensified CCD camera for high spatial and temporal sampling of fluorescent signals and combine it with non-contact detection geometries, concurrent reflectance imaging and high-resolution three-dimensional boundary extraction. Furthermore, we aim at developing fast reconstruction schemes, appropriate for the large data-sets acquired and for non-contact geometries, in order to resolve and quantify fluorochrome concentration, activation and fluorescence lifetime. FMT is one of the few truly enabling technologies for molecular imaging since it capitalizes on signal amplification and fluorescence activation (molecular switches or beacons), high detection sensitivity and chemical specificity. Furthermore, it uses stable reporters, non-ionizing radiation and can simultaneously image multiple targets by spectral differentiation. Recent studies have also shown the potential of using this technology to interrogate human tissues. FMT is expected to enhance our ability to study molecular pathways, perform early detection of disease, real time molecular medicine and ultimately patient-tailored treatment.